1. Field of the Invention
The present invention relates to an electrophoretic apparatus for separating and analyzing a nucleic acid, a protein and the like by using an electrophoretic method, and in particular, to a fluorescent detection technique of an electrophoretic apparatus.
2. Description of the Related Art
A capillary electrophoretic apparatus is disclosed in Japanese Patent Application Laid-Open (JP-A) No. 2004-144479. The capillary electrophoretic apparatus uses an electrophoretic method, in which a capillary composed of a quartz tube and a polymer film covering the quartz tube is used, in order to determine the base sequence and the base length of a DNA. A sample including the DNA to be measured is injected into a separation medium, which is made of polyacrylamide and the like and held in the quartz capillary, and a voltage is applied to both ends of the capillary. A DNA composite contained in the sample moves within the capillary and is separated according to the molecular weight, and DNA bands are generated within the capillary. A fluorescent dye is applied onto each of the DNA bands, and the fluorescent dye develops color by laser beam irradiation. Then, the fluorescent dye is read out by a fluorescence measurement unit so as to determine the sequence of the DNAs. Separation and analysis of the protein can be performed in the same manner so as to examine the structure of the protein.
A method of irradiating a light beam onto a sample, which is disclosed in JP-A No. 2004-144479, is as follows. That is, a laser beam is irradiated onto one capillary, which is located at one side end of a capillary array composed of a plurality of capillaries arranged on a flat substrate, or two capillaries, which are located at both side ends of the capillary array, and the laser beam sequentially propagates to adjacent capillaries so as to traverse the capillary array. In addition, a fluorescent detection method is as follows. That is, an image of a laser beam irradiation unit located on the capillary array is formed on a two-dimensional CCD by a condensing lens, a transmissive diffraction grating, and an imaging lens. Of the two axes on the two-dimensional CCD, one is an axis on which emission points of the plurality of capillaries are arrayed, and the other orthogonal to the one is a wavelength dispersion axis made due to the transmissive diffraction grating. In this way, an emission spectrum output from each of the capillaries is formed on the two-dimensional CCD.
In a conventional capillary electrophoretic apparatus, excitation light is irradiated onto a capillary, fluorescent light which is output from a DNA band that moves within the capillary is dispersed according to the wavelength by a diffraction grating, and the wavelength-dispersed fluorescent light is detected by a two-dimensional optical detector, thereby obtaining an emission spectrum.
However, in the conventional capillary electrophoretic apparatus, when the electrophoretic direction (that is, the movement direction of a DNA band in an irradiation region of the excitation light) and the wavelength dispersion direction of the fluorescent light are the same, the emission spectrum detected by the two-dimensional optical detector substantially changes over time. As a result, the analysis precision becomes worse.
In other words, when the electrophoretic direction and the wavelength dispersion direction of the fluorescent light are the same, since the wavelength-dispersed fluorescent light moves in the wavelength dispersion direction while the DNA band passes through the irradiation region of the excitation light, a signal obtainable by the two-dimensional detector changes. Accordingly, at a time when the DNA band passes through the excitation light, the wavelength of an emission spectrum obtainable by the two-dimensional detector substantially changes over time.
In the electrophoretic analysis, a plurality of fluorescent dyes is used and each of the fluorescent dyes corresponds to each of the four kinds of bases. When the emission spectrum substantially changes over time, it is difficult for the observed emission spectrums to completely correspond to various fluorescent dyes or various bases. That is, when each base corresponds to each component of the emission spectrum, a residual component (quasi peak) that does not correspond to a base is generated, which causes the analysis precision to deteriorate.